Method to enhance hemodynamic stability using oxygen carrying compositions

ABSTRACT

The present invention relates to methods for enhancing the hemodynamic stability of an individual undergoing surgery by administering a composition comprising a hemoglobin-based oxygen carrier. In one embodiment, the present invention relates to the use of polyalkylene oxide modified hemoglobins with reduced cooperativity and a high oxygen affinity to enhance oxygen offloading as a preventative measure to avoid hemodynamic stability-related complications during surgery.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.12/260,945, filed Oct. 29, 2008 now U.S. Pat. No. 7,622,439, which is acontinuation of U.S. Ser. No. 11/218,006, filed Aug. 31, 2005 nowabandoned, which claims priority under 35 U.S.C. §119(e) to U.S. Ser.No. 60/605,816, filed Aug. 31, 2004.

TECHNICAL FIELD

The present invention relates to methods for enhancing the hemodynamicstability of an individual undergoing surgery by administering acomposition comprising a hemoglobin-based oxygen carrier. In oneembodiment, the present invention relates to the use of polyalkyleneoxide modified hemoglobins with reduced cooperativity and a high oxygenaffinity to enhance oxygen offloading as a preventative measure to avoidhemodynamic stability-related complications during surgery.

BACKGROUND OF THE INVENTION

The blood is the means for delivering oxygen and nutrients and removingwaste products from the tissues. The blood is composed of plasma inwhich red blood cells (RBCs or erythrocytes), white blood cells (WBCs),and platelets are suspended. Red blood cells comprise approximately 99%of the cells in blood, and their principal function is the transport ofoxygen to the tissues and the removal of carbon dioxide therefrom.

The left ventricle of the heart pumps the blood through the arteries andthe smaller arterioles of the circulatory system. The blood then entersthe capillaries, where the majority of the delivery of oxygen, exchangeof nutrients and extraction of cellular waste products occurs. (See,e.g., A. C. Guyton, “Human Physiology And Mechanisms Of Disease” (3rd.ed.; W. B. Saunders Co., Philadelphia. Pa.), pp. 228-229 (1982)).Thereafter, the blood travels through the venules and veins in itsreturn to the right atrium of the heart. Though the blood that returnsto the heart is oxygen-poor compared to that which is pumped from theheart, when at rest, the returning blood still contains about 75% of theoriginal oxygen content.

The reversible oxygenation function (i.e., the delivery of oxygen) ofRBCs is carried out by the protein hemoglobin. In mammals, hemoglobinhas a molecular weight (MW) of approximately 64,000 Daltons and iscomposed of about 6% heme and 94% globin. In its native form, itcontains two pairs of subunits (i.e., it is a tetramer), each containinga heme group and a globin polypeptide chain. In aqueous solution,hemoglobin is present in equilibrium between the tetrameric (MW 64,000Daltons) and dimeric (MW 32,000 Daltons) forms. Outside of the RBC, thedimers are prematurely excreted by the kidney (plasma half-life ofapproximately 2-4 hours). Along with hemoglobin, RBCs contain stroma(the RBC membrane), which comprises proteins, cholesterol, andphospholipids.

Due to the demand for blood products in hospitals and other settings,extensive research has been directed at the development of bloodsubstitutes. A “blood substitute” is a blood product that is capable ofcarrying and supplying oxygen to the tissues. Hemoglobin-based oxygencarriers (HBOCs) are blood substitutes containing hemoglobins. HBOCshave a number of uses, including replacing blood lost during surgicalprocedures and following acute hemorrhage, and for resuscitationprocedures following traumatic injury. Essentially, HBOCs can be usedfor any purpose in which banked blood is currently administered topatients. (See, e.g., U.S. Pat. Nos. 4,001,401 to Bonson et al., and4,061,736 to Morris et al.)

The development of HBOCs is especially important, given the fact thatthe current human blood supply is limited. For this reason, human bloodis normally only used in circumstances when it is medically necessary.This usually means that human blood is not appropriate for prophylacticuse, such as “blood doping” (i.e. administering whole blood for thepurpose of enhancing performance by increasing the oxygen carryingcapacity of the blood). Accordingly, neither the use of whole blood norHBOCs for prophylactic indications are widespread, and in most cases areconsidered to be a somewhat questionable practice.

The administration of HBOCs to patients prior to surgery has beenpreviously suggested in combination with the removal of autologous bloodfrom the patients (i.e. acute normovolemic hemodilution or “ANH”) whichcould be returned later in the procedure, if needed, or after surgery.See, for example, PCT WO 98/37909. Such patients are considered hereinnot to be “normovolemic” at the time of surgery. However, this proceduredoes not address the need for prophylactic measures to avoid thedetrimental primary effects of surgical procedures such as hemodynamicstability, and only addresses the secondary effects of blood lossassociated with surgery.

Enhancing hemodynamic stability during surgery requiring generalanesthesia is important for two fundamental reasons. First, hemodynamicinstability caused by blood loss or other factors can lead to tissuedamage and even death. For example, hemorrhagic hypotension andanaphylactic shock are conditions which result from significant bloodloss leading to reduced tissue oxygenation. For patients with suchmedical conditions, it is desirable and often critical for theirsurvival to stabilize their blood pressure and to increase the amount ofoxygen provided to body tissues by their circulatory systems.

Second and most importantly, hemodynamic instability, even minor andtransient, may affect a patient's post-surgical recovery. Thisinstability may occur anywhere throughout the body, and is oftenmanifested as a “hypotensive event,” which is usually recorded as adecrease in blood pressure. Such events can occur as a result offluctuations of localized hemodynamic properties during generalanesthesia, even when there is no loss of blood. These events can causecognitive damage and other complications that exacerbate recoveryfollowing surgery. For example, elderly patients undergoing invasivesurgical procedures such as hip replacements would benefit from anyprophylactic treatment that would enhance their hemodynamic stabilityduring surgery. In addition, such patients are often not suitablecandidates for ANH, and enhancing their hemodynamic stability would beexpected to lessen their need for transfusions using donor blood.

The use of plasma expanders and volume replacements to maintainhemodynamic stability is widespread. However, these non-oxygen carryingsolutions only dilute the oxygen capacity of the blood, even withoutconcomitant ANH, and may actually cause hemodynamic instability in someinstances. In addition to plasma expanders and volume replacements,crystalloid solutions have also been suggested for use in maintaininghemodynamic stability. However, the administration of these solutionsmay result in excessive water retention and edema, which can also causefluctuations in hemodynamic properties.

Accordingly, there is a need for methods to enhance hemodynamicstability which may result in transient hypotensive events that do notdiminish the blood's inherent oxygen carrying capacity. In accordancewith this goal, the present invention relates to a method of enhancinghemodynamic stability by administering compositions comprisinghemoglobin-based oxygen carriers such as specially formulatedpolalkylene oxide modified hemoglobins.

SUMMARY OF THE INVENTION

The present invention relates to the use of a composition in thetreatment of a normovolemic subject undergoing surgery to enhancehemodynamic stability. In one embodiment, the invention relates to amethod for enhancing hemodynamic stability of a normovolemic subjectundergoing surgery comprising: a) administering a composition containinga hemoglobin-based oxygen carrier (HBOC) with an oxygen affinity higherthan whole blood to the subject in connection with the surgery; and b)monitoring the hemodynamic stability of the patient. Such administrationmay occur prior to, during or after surgery, or any combination thereof.In addition, the hemodynamic stability may be measured before, during orafter surgery, or any combination thereof. In addition, monitoring thehemodynamic stability of the patient may take many forms, such asmonitoring the blood pressure of a patient. In one embodiment,hemodynamic stability is characterized by systolic pressure remainingabove 90 mm Hg.

The HBOC of the present invention may take many forms, such aspolyalkylene oxide modified hemoglobin, which may be obtained fromnatural or synthetic sources, including recombinant sources. In additionthe source of hemoglobin may be from humans or other non-human animals.

In another embodiment of the present invention, the has an oxygenaffinity greater than twice that of whole blood, which may include HBOCswith P50s between 4 to 15.

Other aspects of the invention are described throughout thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the FPLC chromatogram of MalPEG-Hb and stroma freehemoglobin (SFH).

FIG. 2 depicts oxygen equilibrium curves for MalPEG-Hb, SFH and wholeblood.

FIG. 3 depicts the survival rate of rats after administration of varioustest solutions.

FIG. 4 depicts the urine output of patients receiving MalPEG-Hb orRinger's Lactate.

FIG. 5 depicts vital signs of patients receiving MalPEG-Hb prior toanesthesia.

FIG. 6 depicts the percentage of patients receiving MalPEG-Hb or aplacebo that exhibit hypotensive events during surgery.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for enhancing the hemodynamicstability of an individual undergoing surgery by administering acomposition comprising a hemoglobin-based oxygen carrier. In oneembodiment, the present invention relates to the use of polyalkyleneoxide modified hemoglobins with reduced cooperativity and a high oxygenaffinity to enhance oxygen offloading as a preventative measure to avoidhemodynamic stability-related complications during surgery.

To facilitate understanding of the invention set forth in the disclosurethat follows, a number of terms are defined below.

DEFINITIONS

The term “hemoglobin” refers generally to the protein contained withinred blood cells that transports oxygen. Each molecule of hemoglobin has4 subunits, 2α chains and 2β chains, which are arranged in a tetramericstructure. Each subunit also contains one heme group, which is theiron-containing center that binds oxygen. Thus, each hemoglobin moleculecan bind 4 oxygen molecules.

The term “modified hemoglobin” includes, but is not limited to,hemoglobin altered by a chemical reaction such as intra- andinter-molecular cross-linking, genetic manipulation, polymerization,and/or conjugation to other chemical groups (e.g., polyalkylene oxides,for example polyethylene glycol, or other adducts such as proteins,peptides, carbohydrates, synthetic polymers and the like). In essence,hemoglobin is “modified” if any of its structural or functionalproperties have been altered from its native state. As used herein, theterm “hemoglobin” by itself refers both to native, unmodified,hemoglobin, as well as modified hemoglobin.

The term “surface-modified hemoglobin” is used to refer to hemoglobindescribed above to which chemical groups such as dextran or polyalkyleneoxide have been attached, most usually covalently. The term “surfacemodified oxygenated hemoglobin” refers to hemoglobin that is in the “R”state when it is surface modified.

The term “stroma-free hemoglobin” refers to hemoglobin from which allred blood cell membranes have been removed.

The term “methemoglobin” refers to an oxidized form of hemoglobin thatcontains iron in the ferric state and cannot function as an oxygencarrier.

The term “MalPEG-Hb” refers to hemoglobin to which malemidyl-activatedPEG has been conjugated. Such MalPEG may be further referred to by thefollowing formula:Hb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—O—CH₃)_(m)  Formula Iwhere Hb refers to tetrameric hemoglobin, S is a surface thiol group, Yis the succinimido covalent link between Hb and Mal-PEG, R is absent oris an alkyl, amide, carbamate or phenyl group (depending on the sourceof raw material and the method of chemical synthesis), [O—CH₂—CH₂]_(n)are the oxyethylene units making up the backbone of the PEG polymer,where n defines the length of the polymer (e.g., MW=5000), and O—CH₃ isthe terminal methoxy group.

The term “plasma expander” refers to any solution that may be given to asubject to increase plasma volume.

The term “oxygen carrying capacity,” or simply “oxygen capacity,” of ablood substitute refers to its capacity to carry oxygen, but does notnecessarily correlate with the efficiency in which it delivers oxygen.Oxygen carrying capacity of a hemoglobin-containing blood substitute isgenerally calculated from hemoglobin concentration, since it is knownthat each gram of hemoglobin binds 1.34 ml of oxygen. Thus, thehemoglobin concentration in g/dl multiplied by the factor 1.34 yieldsthe oxygen capacity in nil/dl. Hemoglobin concentration can be measuredby any known method, such as by using the β-Hemoglobin Photometer(HemoCue, Inc., Angelholm, Sweden.) Similarly, oxygen capacity can bemeasured by the amount of oxygen released from a sample of hemoglobin orblood by using, for example, a fuel cell instrument (e.g., Lex-O₂-Con,Lexington Instruments, Waltham, Mass.)

The term “oxygen affinity” refers to the avidity with which an oxygencarrier such as hemoglobin binds molecular oxygen. This characteristicis defined by the oxygen equilibrium curve which relates the degree ofsaturation of hemoglobin molecules with oxygen (Y axis) with the partialpressure of oxygen (X axis). The position of this curve is denoted bythe value, P50, the partial pressure of oxygen at which the oxygencarrier is half-saturated with oxygen, and is inversely related tooxygen affinity. Hence the lower the P50, the higher the oxygenaffinity. The oxygen affinity of whole blood (and components of wholeblood such as red blood cells and hemoglobin), as well as any oxygencarrier, can be measured by a variety of methods known in the art. (See,e.g., Winslow et at, J. Biol. Chem. 252(7):2331-37 (1977)). Oxygenaffinity may also be determined using a commercially available HEMOX™Analyzer (TCS Scientific Corporation, New Hope, Pa.). (See, e.g.,Vandegriff and Shrager in “Methods in Enzymology” (Everse et al., Eds.)232:460 (1994)).

The term “oxygen-carrying component” refers broadly to a substancecapable of carrying oxygen in the body's circulatory system anddelivering at least a portion of that oxygen to the tissues. Inpreferred embodiments, the oxygen-carrying component is native ormodified hemoglobin, and is also referred to herein as a“hemoglobin-based oxygen carrier,” or “HBOC.”

The term “hemodynamic parameters” refers broadly to measurementsindicative of blood pressure, flow and volume status, including directmeasurements such as blood pressure, cardiac output, right atrialpressure, left ventricular end diastolic pressure, as well as indirectmeasurements of tachycardia, ischemia, bradycardia, conduction problems,fluid balance, weight, ICU time and kidney function.

The term “crystalloid” refers to small molecules (usually less than 10Å) such as salts, sugars, and buffers. Unlike colloids, crystalloids donot contain any oncotically active components and equilibrate in betweenthe circulation and interstitial spaces very quickly.

The term “colloid” (in contrast to “crystalloid”) refers to largermolecules (usually greater than 10 Å) that equilibrate across biologicalmembranes depending on their size and charge and includes proteins suchas albumin and gelatin, as well as starches such as pentastarch andhetastarch.

The term “colloid osmotic pressure” refers to the pressure exerted by acolloid to equilibrate fluid balance across a membrane.

The term “stable to autooxidation” refers to the ability of a HBOC tomaintain a low rate of autoxidation. A HBOC is considered stable at 24°C. if the methemoglobin/total hemoglobin ratio does not increase morethan 2% after 10 hours at room temperature (approximately 24° C.) Forexample, if the rate of autoxidation is 0.2 hr⁻¹, then if the initialpercentage of methemoglobin is 5%, and the HBOC would be consideredstable at room temperature for 10 hours if this percentage did notincrease above 7%.

The term “methemoglobin/total hemoglobin ratio” refers to the ratio ofoxidized hemoglobin to total hemoglobin.

The term “mixture” refers to a mingling together of two or moresubstances without the occurrence of a reaction by which they would losetheir individual properties; the term “solution” refers to a liquidmixture; the term “aqueous solution” refers to a solution that containssome water and may also contain one or more other liquid substances withwater to form a multi-component solution; the term “approximately”refers to the actual value being within a range, e.g. 10%, of theindicated value.

The term “polyethylene glycol” or “PEG” refers to liquid or solidpolymers of the general chemical formula H(OCH₂CH₂)_(n)OH, where n isgreater than or equal to 4, and variants thereof, such as PEG that isactivated, substituted and/or unsubstituted.

The term “perfusion” refers to the flow of fluid to tissues and organsthrough arteries and capillaries.

The term “hemodynamic stability” refers to stable functioning in themechanics of circulation, i.e. the stability over a given period of timeof any hemodynamic parameter.

The term “hypotensive events” is characterized by or due to localized orgeneralized hypotension, i.e. a lowering of blood pressure, which canfurther be defined quantitatively as a systolic pressure less than 90mmHg or a reduction of blood pressure below 75% of the baseline value.

The meaning of other terminology used herein should be easily understoodby someone of reasonable skill in the art.

The Nature of Oxygen Delivery and Consumption

Although the successful use of the compositions and methods of thepresent invention do not require comprehension of the underlyingmechanisms of oxygen delivery and consumption, basic knowledge regardingsome of these putative mechanisms may assist in understanding thediscussion that follows. It has generally been assumed that thecapillaries are the primary conveyors of oxygen to the tissue. However,regarding tissue at rest, current findings indicate that there isapproximately an equipartition between arteriolar and capillary oxygenrelease. That is, hemoglobin in the arterial system is believed todeliver approximately one third of its oxygen content in the arteriolarnetwork and one-third in the capillaries, while the remainder exits themicrocirculation via the venous system.

The arteries and arterioles themselves are sites of oxygen utilization.For example, the artery wall requires energy to effect regulation ofblood flow through contraction against vascular resistance. Thus, thearterial wall is normally a significant site for the diffusion of oxygenout of the blood. However, current oxygen-delivering compositions (e.g.,HBOCs) may release too much of their oxygen content in the arterialsystem, and thereby induce an autoregulatory reduction in capillaryperfusion. Accordingly, the efficiency of oxygen delivery of a bloodsubstitute may actually be hampered by having too much oxygen or too lowan oxygen affinity.

The rate of oxygen consumption by the vascular wall, i.e., thecombination of oxygen required for mechanical work and oxygen requiredfor biochemical synthesis, can be determined by measuring the gradientat the vessel wall. See, e.g., Winslow, et al., in “Advances in BloodSubstitutes” (1997), Birkhauser, Ed., Boston, Mass., pages 167-188.Present technology allows accurate oxygen partial pressure measurementsin a variety of vessels. The measured gradient is directly proportionalto the rate of oxygen utilization by the tissue in the region of themeasurement. Such measurements show that the vessel wall has a baselineoxygen utilization which increases with increases in inflammation andconstriction, and is lowered by relaxation.

The vessel wall gradient is inversely proportional to tissueoxygenation. Vasoconstriction increases the oxygen gradient (tissuemetabolism), while vasodilation lowers the gradient. Higher gradientsare indicative of the fact that more oxygen is used by the vessel wall,while less oxygen is available for the tissue. The same phenomenon isbelieved to be present throughout the microcirculation.

The Relationship Between Vasoconstriction and Oxygen Affinity

The rationale for developing an HBOC with high oxygen affinity is based,in part, on past studies using cell-free hemoglobins as alternatives tored blood cell transfusions. Some of the physiological effects of thesesolutions remain incompletely understood. Of these, perhaps the mostcontroversial is the propensity to cause vasoconstriction, which may bemanifest as hypertension in animals and man (Amberson, W., “Clinicalexperience with hemoglobin-saline solutions,”. Science 106: 117-117(1947)) (Keipert, P., A. Gonzales, C. Gomez, V. Macdonald, J. Hess, andR. Winslow, “Acute changes in systemic blood pressure and urine outputof conscious rats following exchange transfusion withdiaspirin-crosslinked hemoglobin solution,” Transfusion 33: 70 1-708,(1993)). Human hemoglobin crosslinked between α chains withbis-dibromosalicyl-fumarate (ααHb) was developed by the U.S. Army as amodel red cell substitute, but was abandoned by the Army afterdemonstration of severe increases in pulmonary and systemic vascularresistance (Hess, J., V. Macdonald, A. Murray, V. Coppes, and C. Gomez,“Pulmonary and systemic hypertension after hemoglobin administratio,”Blood 78: 356A (1991)). A commercial version of this product was alsoabandoned after a disappointing Phase III clinical trial (Winslow, R. M.“αα-Crosslinked hemoglobin: Was failure predicted by preclinicaltesting?” Vox sang 79: 1-20 (2000).

The most commonly advanced explanation for the vasoconstriction producedby cell-free hemoglobin is that it readily binds the endothelium-derivedrelaxing factor, nitric oxide (NO). In fact, recombinant hemoglobinswith reduced affinity for NO have been produced which appear to be lesshypertensive in top-load rat experiments (Doherty, D. H., M. P. Doyle,S. R. Curry, R. J. Vali, T. J. Fattor, J. S. Olson, and D. D. Lemon,“Rate of reaction with nitric oxide determines the hypertensive effectof cell-free hemoglobin,” Nature Biotechnology 16: 672-676 (1998))(Lemon, D. D., D. H. Doherty, S. R. Curry, A. J. Mathews, M. P. Doyle,T. J. Fattor, and J. S. Olson, “Control of the nitric oxide-scavengingactivity of hemoglobin,” Art Cells, Blood Subs., and Immob. Biotech24:378 (1996)).

However, studies suggest that NO binding may not be the only explanationfor the vasoactivity of hemoglobin. It has been found that certain largehemoglobin molecules, such as those modified with polyethylene glycol(PEG), were virtually free of the hypertensive effect, even though theirNO binding rates were identical to those of the severely hypertensiveααHb (Rohlfs, R. J., E. Bruner, A. Chiu, A. Gonzales, M. L. Gonzales, D.Magde, M. D. Magde, K. D. Vandegriff, and R. M. Winslow, “Arterial bloodpressure responses to cell-free hemoglobin solutions and the reactionwith nitric oxide,” J BioI Chem 273: 12128-12134 (1998)). Furthermore,it was found that PEG-hemoglobin was extraordinarily effective inpreventing the consequences of hemorrhage when given as an exchangetransfusion prior to hemorrhage (Winslow, R. M., A. Gonzales, M.Gonzales, M. Magde, M. McCarthy, R. J. Rohlfs, and K. D. Vandegriff“Vascular resistance and the efficacy of red cell substitutes,” J ApplPhysiol 85: 993-1003 (1998)).

This protective effect correlated with the lack of hypertension,suggesting that vasoconstriction is responsible for the disappointingperformance of many of the hemoglobin-based products studied to date.Based on these observations, a hypothesis was developed to explainvasoconstriction, as an alternative, or possibly in addition to, theeffect of NO binding. Although not wishing to be bound by any particulartheory, it is believed that a substantial component of hemoglobin'svasoactive effect is a reflexive response to the diffusion of hemoglobinin the cell-free space. This hypothesis was tested in an in vitrocapillary system, and it was demonstrated that PEG-hemoglobin, which hasa reduced diffusion constant, transferred O₂ in a manner very similar tothat of native red blood cells (McCarthy, M. R., K. D. Vandegriff, andR. M. Winslow, “The role of facilitated diffusion in oxygen transport bycell-free hemoglobin: Implications for the design of hemoglobin-basedoxygen carriers,” Biophysical Chemistry 92: 103-117 (2001)). Oxygenaffinity would be expected to play a role in its facilitated diffusionby hemoglobin in the plasma space, since the change in saturation fromthe hemoglobin to the vessel wall is a determinant of the diffusiongradient of the hemoglobin itself.

Oxygen affinity of cell-free hemoglobin may play an additional role inthe regulation of vascular tone, since the release of O₂ to vessel wallsin the arterioles will trigger vasoconstriction (Lindbom, L., R. Tuma,and K. Arfors, “Influence of oxygen on perfusion capillary density andcapillary red cell velocity in rabbit skeletal muscle,” Microvasc Res19:197-208 (1980)). In the hamster skinfold, the PO₂ in such vessels isin the range of 20-40 Torr, where the normal red cell oxygen equilibriumcurve is steepest (Intaglietta, M., P. Johnson, and R. Winslow,“Microvascular and tissue oxygen distribution,” Cardiovasc Res 32:632-643 (1996)). Thus from a theoretical point of view, it may beimportant for the P50 of cell-free hemoglobin to be lower than that ofred cells (i.e., higher O₂ affinity), in order to prevent release of O₂in arteriolar regulatory vessels.

Oxygen Offloading

In addition to oxygen affinity, the oxygen binding propertiesthemselves, i.e. the cooperativity and allosteric effects, may play acrucial role in the oxygen offloading capabilities of HBOCs. It has beenobserved that polyalkylene binding to hemoglobin results in a general“tightening” of the globin structure. This is attributed to the osmoticeffects of having a hydrophilic shell surrounding the hemoglobin, andmay also depend on the nature and location of the linking groups used toattach the polyalkylene oxide. Most conventional wisdom is that thedesign of HBOCs should mimic the characteristics of native hemoglobin.However, it has unexpectedly been found that perturbation of thequaternary conformation of the hemoglobin can have advantages,particularly in the context of oxygen offloading.

A protein is considered to be “allosteric” if its characteristics changeas a result of binding to an effector molecule, i.e. a ligand, at itsallosteric site. In the case of hemoglobin, the ligand is oxygen. Eachsubunit of the hemoglobin tetramer is capable of binding one oxygenmolecule. Each subunit also exists in one of two conformations—tensed(T) or relaxed (R). In the R state, it can bind oxygen more readily thanin the T state.

Hemoglobin exhibits a concerted effect, or cooperativity, amongindividual subunits binding oxygen. The binding of oxygen to one subunitinduces a conformational change in that subunit that causes theremaining active sites to exhibit an enhanced oxygen affinity.Accordingly, each sequential oxygen molecule that binds to thehemoglobin molecule attaches more readily than the one before, until thehemoglobin molecule has achieved the R, or “liganded” state, with fourattached oxygen molecules.

In the reverse, native hemoglobin exhibits a concerted effect in termsof its efficiency to release oxygen. The first molecule is more tightlyattached and takes more energy to be “offloaded” then the next one, andso on. Accordingly, the conventional teachings towards the design ofblood substitutes that mimic the cooperativity of native hemoglobin mayadversely affect its ability to release oxygen once bound.

The present invention relates to the finding that HBOCs with less, notmore, cooperativity than native hemoglobin are unexpectedly more usefulin applications involving oxygen offloading. Accordingly, a compositionfor use in enhancing hemodynamic stability must be able to readilyrelease its oxygen once it arrives at its target location, but must notcontain too much oxygen releasing capacity to avoid vasoconstrictiveeffects. In summary, the ideal composition for prophylactic treatment inconnection with a surgical procedure to enhance hemodynamic stabilitynecessarily contains a modified hemoglobin with less cooperativity thannative hemoglobin, but which is formulated in a composition with a muchhigher oxygen affinity (e.g. less than half the P50) when compared towhole blood.

Oxygen-Carrying Component

In preferred embodiments, the oxygen carrier (i.e., the oxygen-carryingcomponent) is a hemoglobin-based oxygen carrier, or HBOC. The hemoglobinmay be either native (unmodified); subsequently modified by a chemicalreaction such as intra- or inter-molecular cross-linking,polymerization, or the addition of chemical groups (e.g., polyalkyleneoxides, or other adducts); or it may be recombinantly engineered. Humanalpha- and beta-globin genes have both been cloned and sequenced.Liebhaber, et al, P.N.A.S. 77: 7054-7058 (1980); Marotta, et al., J.Biol. Chem. 353: 5040-5053 (1977) (beta-globin cDNA). In addition, manyrecombinantly produced modified hemoglobins have now been produced usingsite-directed mutagenesis, although these “mutant” hemoglobin varietieswere reported with high oxygen affinities. See, e.g., Nagai, et al.,P.N.A.S., 82:7252-7255 (1985).

The HBOCs that are used in the practice of the present invention haveoxygen affinities higher than normal whole blood (from the same animalsource as the subject, which may not necessarily be a human), which isalso expressed as a P50 lower than that of whole blood. Whole blood isgenerally considered to have a P50 approximating 28 torr. In oneembodiment, the HBOCs of the present invention have a P50 less than halfthat of whole blood, which is considered to be a “high oxygen affinity.”Such high oxygen affinity HBOCs may have a P50 of 4-15, such as 10, 7,etc.

The present invention is not limited by the source of the hemoglobin.For example, the hemoglobin may be derived from animals and humans.Preferred sources of hemoglobin for certain applications are humans,cows and pigs, as well as non-mammalian sources, such as annelids,reptiles, etc. In addition, hemoglobin may be produced by other methods,including chemical synthesis and recombinant techniques. The hemoglobincan be added to the blood product composition in free form, or it may beencapsulated in a vesicle, such as a synthetic particle, microballoon orliposome. The preferred oxygen-carrying components of the presentinvention should be stroma free and endotoxin free. Representativeexamples of oxygen-carrying components are disclosed in a number ofissued United States patents, including U.S. Pat. No. 4,857,636 to Hsia;U.S. Pat. No. 4,600,531 to Walder, U.S. Pat. No. 4,061,736 to Morris etal.; U.S. Pat. No. 3,925,344 to Mazur; U.S. Pat. No. 4,529,719 to Tye;U.S. Pat. No. 4,473,496 to Scannon; 4,584,130 to Bocci et al; U.S. Pat.No. 5,250,665 to Kluger, et al.; U.S. Pat. No. 5,028,588 to Hoffman etal and U.S. Pat. No. 4,826,811 and U.S. Pat. No. 5,194,590 to Sehgal etal.

In addition to the aforementioned sources of hemoglobin, it has recentlybeen found that horse hemoglobin may have certain advantages as theoxygen carrying component in the compositions of the present invention.One advantage is that commercial quantities of horse blood are readilyavailable from which horse hemoglobin can be purified. Anotherunexpected advantage is that horse hemoglobin exhibits chemicalproperties that may enhance its usefulness in the blood substitutes ofthe present invention.

Previous reports have indicated that horse hemoglobin auto-oxidizes tomethemoglobin faster than human hemoglobin, which would make it lessdesirable as a blood substitute component. See, e.g., J. G. McLean andI. M. Lewis, Research in Vet. Sci., 19:259-262 (1975). In order tominimize auto-oxidation, McLean and Lewis used a reducing agent,glutathione, after red blood cell lysis. However, the hemoglobin that isused to prepare the compositions of the present invention, regardless ofwhether the source of hemoglobin is human or horse, do not require theuse of reducing agents to prevent auto-oxidation after red blood celllysis.

More recently, it has been reported that horse hemoglobin has an oxygenaffinity that is different from that of human hemoglobin. See, e.g., M.Mellegrini, et al. Eur. J. Biochem., 268: 3313-3320 (2001). Such adifference would discourage the selection of horse hemoglobin to prepareblood substitutes that mimic human hemoglobin. However, whenincorporated into the compositions of the present invention, nosignificant difference (less than 10%) in oxygen affinity between humanand horse hemoglobin-containing conjugates is observed. Accordingly,contrary to these seemingly undesirable properties, in the compositionsof the present invention, horse hemoglobin may be equivalent to humanhemoglobin.

For use in the present invention, the HBOC has an oxygen affinity thatis greater than whole blood, and preferably twice that of whole blood,or alternatively, greater than that of stroma-free hemoglobin (SFH),when measured under the same conditions. In most instances, this meansthat the HBOC in the blood substitute will have a P50 less than 10, andmore preferably less than 7. In the free state, SFH has a P50 ofapproximately 15 torr, whereas the P50 for whole blood is approximately28 torr. It has previously been suggested that increasing oxygenaffinity, and thereby lowering the P50, may enhance delivery of oxygento tissues, although it was implied that a P50 lower than that of SFHwould not be acceptable. See Winslow, et al., in “Advances in BloodSubstitutes” (1997), Birkhauser, ed., Boston, Mass., at page 167, andU.S. Pat. No. 6,054,427. This suggestion contradicts the widely heldbelief that modified hemoglobins for use as blood substitutes shouldhave lower oxygen affinities, and should have P50s that approximate thatof whole blood. Hence, many researchers have used pyridoxyl phosphate toraise the P50 of SFH from 10 to approximately 20-22, since pyridoxylatedhemoglobin more readily releases oxygen when compared to SFH.

There are many different scientific approaches to manufacturing HBOCswith high oxygen affinity (i.e. those with P50s less than SFH). Forexample, studies have identified the amino acid residues that play arole in oxygen affinity, such as β-93 cysteine, and thus site-directedmutagenesis can now be easily carried out to manipulate oxygen affinityto the desired level. See, e.g., U.S. Pat. No. 5,661,124. Many otherapproaches are discussed in U.S. Pat. No. 6,054,427.

Hemoglobin-Associated Toxicity

Hemoglobin is known to exhibit autooxidation when it reversibly changesfrom the ferrous (Fe²⁺) to the ferric (Fe³⁺) or methemoglobin form. Whenthis happens, molecular oxygen dissociates from the oxyhemoglobin in theform a superoxide anion (O²⁻). This also results in destabilization ofthe heme-globin complex and eventual denaturation of the globin chains.Both oxygen radical formation and protein denaturation are believed toplay a role in in vivo toxicity of HBOCs (Vandegriff, K. D BloodSubstitutes, Physiological Basis of Efficacy, pages 105-130, Winslow etal., ed., Birkhauser, Boston, Mass. (1995).)

With most HBOCs, there is a negative correlation between oxygen affinityand hemoglobin oxidation, i.e., the higher the oxygen affinity, thelower the rate of autooxidation. However, the effects of differenthemoglobin modifications on oxygen affinity and the rate ofautooxidation are not always predictable. In addition, the optimalbalance between oxygen affinity and autooxidation rate is not wellunderstood.

In one embodiment, the compositions of the present invention containpolyalkylene oxide-Hb conjugates, such as polyethylene glycol-Hbconjugates that exhibit very low rates of autooxidation at roomtemperature. When measured as a rate of oxidation, this value should beas low as possible (i.e., 0.2% per hour of total hemoglobin, morepreferably 0.1% per hour of total hemoglobin, at room temperature for atleast 3 hours, and more preferably at least 10 hours.) Thus, exemplaryHBOCs of the present invention remain stable during administrationand/or storage at room temperature.

Modifications of the Oxygen-Carrying Component

In an exemplary embodiment, the oxygen-carrying component ispolyalkylene oxide (PAO) modified hemoglobin. Suitable PAOs include,inter alia, polyethylene oxide ((CH₂CH₂O)_(n)), polypropylene oxide((CH(CH₃)CH₂O)_(n)) or a polyethylene/polypropylene oxide copolymer((CH₂CH₂O)_(n)—(CH(CH₃)CH₂O)_(n)). Other straight, branched chain andoptionally substituted synthetic polymers that would be suitable in thepractice of the present invention are well known in the medical field.

Most commonly, the chemical group attached to the hemoglobin ispolyethylene glycol (PEG), because of its pharmaceutical acceptabilityand commercial availability. PEGs are polymers of the general chemicalformula H(OCH₂CH₂)_(n)OH, where n is generally greater than or equal to4. PEG formulations are usually followed by a number that corresponds totheir average molecular weight. For example, PEG-200 has an averagemolecular weight of 200 and may have a molecular weight range of190-210. PEGs are commercially available in a number of different forms,and in many instances come preactivated and ready to conjugate toproteins.

An important aspect of exemplary embodiments of the present invention isthat surface modification takes place when the hemoglobin is in theoxygenated or “R” state. This is easily accomplished by allowing thehemoglobin to equilibrate with the atmosphere (or, alternatively, activeoxygenation can be carried out) prior to conjugation. By performing theconjugation to oxygenated hemoglobin, the oxygen affinity of theresultant hemoglobin is enhanced. Such a step is generally regarded asbeing contraindicated, since many researchers describe deoxygenationprior to conjugation to diminish oxygen affinity. See, e.g., U.S. Pat.No. 5,234,903.

Although in many respects the performance of PAO modified hemoglobins isindependent of the linkage between the hemoglobin and the modifier (e.g.PEG), it is believed that the type of linkers such as more rigidunsaturated aliphatic or aromatic C₁ to C₆ linker substituents maychange the manufacturing and/or characteristics of the conjugates whencompared to those that have different linkages, such as more deformablemodes of attachment when compared to the rigid linkers just described.

The number of PEGs to be added to the hemoglobin molecule may vary,depending on the size of the PEG. However, the molecular size of theresultant modified hemoglobin should be sufficiently large to avoidbeing cleared by the kidneys to achieve the desired half-life.Blumenstein, et al., determined that this size is achieved above amolecular weight of 84,000 Daltons. (Blumenstein, et al., in “BloodSubstitutes and Plasma Expanders,” Alan R. Liss, editors, New York,N.Y., pages 205-212 (1978).) Therein, the authors conjugated hemoglobinto dextran of varying molecular weight. They reported that a conjugateof hemoglobin (with a molecular weight of 64,000 Daltons) and dextran(having a molecular weight of 20,000 Daltons) “was cleared slowly fromthe circulation and negligibly through the kidneys,” but increasing themolecular weight above 84,000 Daltons did not alter the clearancecurves. Accordingly, as determined by Blumenstein, et al., it ispreferable that the HBOC have a molecular weight of at least 84,000Daltons.

In one embodiment of the present invention, the HBOC is a “MalPEG-Hb,”which stands for hemoglobin to which malemidyl-activated PEG has beenconjugated. Such MalPEG-Hb may be further referred to by the followingformula:Hb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—O—CH₃)_(m)  Formula Iwhere Hb refers to tetrameric hemoglobin, S is a surface thiol group, Yis the succinimide covalent link between Hb and Mal-PEG, R is absent oris an alkyl, amide, carbamate or phenyl group (depending on the sourceof raw material and the method of chemical synthesis), [O—CH₂—CH₂]_(n)are the oxyethylene units making up the backbone of the PEG polymer,where n defines the length of the polymer (e.g., MW=5000), and O—CH₃ isthe terminal methoxy group.Formulation

The HBOCs of the present invention are formulated by mixing the HBOC andother optional excipients with a suitable aqueous solution, or “diluent”(i.e., one which is pharmaceutically acceptable for intravenousinjection.) Although the concentration of the oxygen carrier in thediluent may vary according to the application, and in particular basedon the expected post-administration dilution, in preferred embodiments,because of the other features of the compositions of the presentinvention that provide for enhanced oxygen delivery and therapeuticeffects, it is usually unnecessary for the concentration of hemoglobinin an HBOC to be above 6 g/dl, and may be between 0.1 to 4 g/dl.

Suitable diluents may also include, inter alia, proteins, glycoproteins,polysaccharides, and other colloids. It is not intended that theseembodiments be limited to any particular diluent. Thus, it is intendedthat the diluent encompass aqueous cell-free solutions of albumin, othercolloids, or other non-oxygen carrying components. In some embodiments,the aqueous solution may have a viscosity of at least 2.5 cP. In otherembodiments, the viscosity of the aqueous solution is between 2.5 and 4cP.

Administration of the compositions described herein may occur at anytime “in connection with surgery”, including as long as a day before,during or a day after surgery.

Clinical Applications

The present invention relates to HBOCs such as PAO modified hemoglobinconjugates that may exhibit less cooperativity than native hemoglobinthat are formulated into compositions with lower P50s than whole blood,which in some embodiments are less than half the P50 of whole blood.Such compositions are useful to enhance hemodynamic stability innormovolemic patients undergoing surgery. They may be administered usingthe same administration parameters that are well known in the art forplasma expanders, volume enhancers and such. Hemodynamic stability maybe monitored before, during or after surgery, or any combinationthereof. Thus, enhancement of hemodynamic stability is not necessarilylimited to the measurement of the hemodynamic properties of the subjectjust during the surgical procedure itself, and may be observed any timepost-administration.

The patient population that may benefit the most from the practice ofthe present invention method is “normovolemic patients undergoingsurgery.” Thus, in one embodiment, the present invention method relatesto the administration of the compositions to patients that have notundergone the extraction of blood during an acute normovolemichemodilution (ANH) treatment. This is based on the finding thatindividuals receiving these compositions in connection with a surgicalprocedure are less likely to require a blood transfusion during or aftersurgery. Hence, the methods of the present invention are, in one aspect,an alternative to ANH.

In another aspect, the present invention method may also result in theindirect benefit of a normovolemic patient undergoing surgery requiringless administration of vasopressors during surgery to raise bloodpressure, because their blood pressure is effectively stabilized duringsurgery due to the prophylactic administration of the HBOC in connectionwith the surgery.

EXAMPLES

A similar version of the following three Examples are disclosed U.S.Patent Application No. 2003/0162693, which published on Aug. 28, 2003.As for all of the Examples, MalPEG-Hb is used as a model HBOC. Theproduction and availability of other HBOCs are well known in thescientific literature, yet the ability to conduct clinical trials inhuman subjects is carefully regulated and impossible to perform usingmultiple forms of HBOCs. Thus, based on the teachings presented hereinit should be well understood to one of skill in the art that the scopeof the present invention is not limited to the use of MalPEG-Hb, andHBOCs with similar characteristics (e.g., a higher oxygen affinity thannative hemoglobin, and in particular PAO modified hemoglobin) would alsobe suitable for use.

Example 1 Production of Stroma-Free Hemoglobin

Step 1: Procurement of Outdated Red Blood Cells

Outdated packed red blood cells are procured from a commercial source,such as the San Diego Blood Bank or the American Red Cross. Preferably,outdated material is received not more than 45 days from the time ofcollection. Packed RBCs (pRBCs) are stored at 4±2° C. until furtherprocessed (1-7 days). All units are screened for viral infection andsubjected to nucleic acid testing prior to use.

Step 2: Pooling of Outdated Blood

Packed red blood cells are pooled into a sterile vessel in a cleanfacility. Packed red blood cell volume is noted, and hemoglobinconcentration is determined using a commercially available co-oximeteror other art-recognized method.

Step 3: Leukodepletion

Leukodepletion (i.e. removal of white blood cells) is carried out usingmembrane filtration. Initial and final leukocyte counts are made tomonitor the efficiency of this process.

Step 4: Cell Separation and Cell Wash

Red blood cells are washed with six volumes of 0.9% sodium chloride. Theprocess is carried out at 4±2° C. The cell wash is analyzed to verifyremoval of plasma components by a spectrophotometric assay for albumin.

Step 5: Red Blood Cell Lysis and Removal of Cell Debris

Washed red blood cells are lysed at least 4 hours or overnight at 4±2°C. with stirring using 6 volumes of water. Lysate is processed in thecold to purify hemoglobin. This is achieved by processing the lysatethrough a 0.16-μm membrane. Purified hemoglobin is collected in asterile depyrogenated vessel. All steps in this process are carried outat 4±2° C.

Step 6: Viral Removal

Viral removal is performed by ultrafiltration at 4±2° C.

Step 7: Concentration and Solvent Exchange

Hemoglobin purified from lysate and ultrafiltration is exchanged intoRinger's lactate (RL) or phosphate-buffered saline (PBS, pH 7.4) using a10 lD membrane. The hemoglobin is then concentrated using the samemembrane to a final concentration of 1.1-1.5 mM (in tetramer). Ten to 12volumes of RL or PBS are used for solvent exchange. This process iscarried out at 4±2° C. The pH of the solution prepared in RL is adjustedto 7.0-7.6.

Step 8: Sterile Filtration

Hemoglobin in PBS or Ringer's lactate (RL) is sterile-filtered through a0.45- or 0.2-μm disposable filter capsule and stored at 4±2° C. beforethe chemical modification reaction is performed.

Other methods for purifying hemoglobin are well known in the art. Inaddition, the use of a reducing agent (e.g. glutathione or anotherthiol-containing reducing agent) to prevent auto-oxidation after celllysis is usually unnecessary.

Example 2 Modification of Stroma Free Hemoglobin

Step 1: Thiolation

Thiolation is carried out using 10-fold molar excess iminothiolane overhemoglobin for 4 hours at 4±2° C. with continuous stirring.

Reaction Conditions:

1 mM hemoglobin (tetramer) in RL (pH 7.0-7.5) or PBS (pH 7.4)

10 mM iminothiolane in RL (pH 7.0-7.5) or PBS (pH 7.4)

The ratio of 1:10 SFH:iminothiolane and reaction timing were optimizedto maximize the number of PEGylated thiol groups and to minimize productheterogeneity.

Step 2: PEGylation of Thiolated Hemoglobin

Thiolated hemoglobin is PEGylated using a 20-fold molar excess of MalPEG (with an alkyl or phenyl linker) based on starting tetramerichemoglobin concentration. The hemoglobin is first allowed to equilibratewith the atmosphere to oxygenate the hemoglobin. The reaction takesplace for 2 hours at 4±2° C. with continuous stirring.

Reaction Conditions:

1 m thiolated hemoglobin in RL or PBS (pH 7.4)

20 mM Mal-PEG in RL or PBS (pH 7.4)

Step 3: Removal of Unreacted Reagents

PEGylated-Hb is processed through a 70-kD membrane to remove excessunreacted reagents or hemoglobin. A 20-volume filtration is carried outto ensure removal of unreacted reagents, which is monitored bysize-exclusion chromatography at 540 nm and 280 nm, The proteinconcentration is diluted to 4 g/dl. The pH is adjusted to 7.3±0.3 using1 N NaOH.

Step 4: Sterile Filtration

The final MalPEG-Hb product is sterile-filtered using a 0.2-μm steriledisposable capsule and collected into a sterile depyrogenated vessel at4±2° C.

Step 5: Formulation of MalPEG-Hb

PEGylated Hb is diluted to 4 g/dl RI, pH adjusted to 7.4±0.2.

Step 6: Sterile Fill

The final blood substitute composition is sterile-filtered (0.2 μm) andaliquoted by weight into sterile glass vials and closed with sterilerubber stoppers with crimped seals in a laminar flow hood and stored at−80° C. until use.

Example 3 Physiochemical Analysis of MalPEG-Hb

Methodology for Physicochemical Analysis

Homogeneity and molecular size of the MalPEG-Hb blood substitute arecharacterized by Liquid Chromatography (LC). Analytical LC is used toevaluate homogeneity of the PEGylated hemoglobin and extent of removalof unreacted Mal-PEG. Absorbance at 540 urn is used to evaluatehemoglobin and resolves PEGylated hemoglobin from unreacted hemoglobinby peak position. Absorbance at 280 nm is used to resolve PEGylatedhemoglobin from free Mal-PEG, which absorbs in the ultraviolet (UV)spectrum due to the ring structures in MalPEG.

Optical spectra are collected using a rapid scanning diode arrayspectrophotometer (Milton Roy 2000 or Hewlett Packard Model 8453) in theSoret and visible regions for analysis of hemoglobin concentration andpercent methemoglobin by multicomponent analysis (Vandegriff K. D., andR. E., Shrager. Evaluation of oxygen equilibrium binding to hemoglobinby rapid-scanning spectrophotometry and singular value decomposition.Meth. Enzymol. 232: 460-485 (1994).

MalPEG-Hb concentration and percentage methemoglobin are determinedusing a co-oximeter. Viscosity is determined using a Rheometer. ColloidOsmotic Pressure is determined using a colloid osmometer. Oxygen bindingparameters are determined from oxygen equilibrium curves.

Exemplary specifications for the blood substitute composition arepresented in Table 1 below:

TABLE 1 Test Specification Hemoglobin concentration (g/dl) 4.2 ± 0.2Methemoglobin (%) <10 pH 7.4 ± 0.4 Conductivity (mS/cm) 12 ± 4 Endotoxin (EU/mL)   <0.5 FPLC retention time (min) 43 ± 3  FPLC peakwidth at half height (min) 6 ± 2 Viscosity (cPs) 2.5 ± 1.0 COP (mmHg) 50± 20 P50(Torr) 6 ± 2 Hill number (at P50) 1.2 ± 0.5 Sterility PassNumber of PEGylated Sites on MalPEG-Hb

For surface modification, the number “m” in Formula I is the parameterthat defines the number of PEG polymers attached to the surface ofhemoglobin.Hb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—O—CH₃)_(m)  Formula I

To determine this number, a dithiopyridine colorimetric assay (AmpulskiR., V. Ayers, and S. Morell. Determination of the reactive sulfhydrylgroups in heme proteins with 4,4′-dipyridinesdisulde. Biocheim. Biophys.Acta 163-169, 1969) is used to measure the number of available thiolgroups on the surface of the Hb tetramer before and after thiolation andthen again after Hb PEGylation. Human hemoglobin contains 2 intrinsicreactive thiol groups at the β93Cys residues, which is confirmed by thedithiopyridine reaction. After thiolation of SFH at a ratio of 1:10SFH:iminothiolane, the number of reactive thiol groups increases from 2to 6 thiols based on the dithiopyridine reaction. After the PEGylationreaction, the number of reactive thiol groups is decreased to 1.3. Thisindicates that there are 4-5 PEGylated sites on MaLPEG-Hb.

Size-Exclusion Chromatography Analysis of MalPEG-Hb Versus SFH

FPLC is performed for analysis of the final MaLPEG-Hb product. Typicalchromatograms are displayed in FIG. 1 for MaLPEG-Hb compared tounmodified SFH. The retention time for SFH is approximately 57 mm. Theretention time for MaLPEG-Hb is approximately 44 Min.

Physical and Chemical Characteristics of MalPEG-Hb

The physical properties of MaIPEG-Hb compared to blood and unmodifiedhuman hemoglobin (SFH) are shown below in Table 2.

TABLE 2 Blood SFH MalPEG-Hb P50(Torr) 28 15 5 N50 (Hill number) 2.9 2.91.2 Bohr effect (ΔLog — −0.46 −0.20 P50/ΔpH) Viscosity (cPs) 4.0 0.9 2.5COP (mm Hg)¹ 27 16 50 MW (kD)² N/A 65 90 Molecular Radius (nm) 4000 3.2²9 ¹Determined at 15 g/dl for whole blood and approximately 4 g/dl forhemoglobin solutions ²Determined by COP measurements and FPLCOxygen Affinity

Hemoglobin-oxygen equilibrium binding curves were measured duringenzymatic oxygen consumption. (Anal Biochem. 256: 107-116, 1998).MalPEG-Hb exhibits a high oxygen affinity (P50=5 mm Hg) and lowcooperativity (n50=1.0-1.4). FIG. 2 shows representative curvescomparing stroma-free hemoglobin (SFH), MalPEG-Hb and blood.

Viscosity

This solution property of MalPEG-Hb is due to the strong interactionbetween polyethylene glycol chains and solvent water molecules. This isbelieved to be an important attribute for a MalPEG-Hb blood substitutefor two reasons: 1) higher viscosity decreases the diffusion constant ofboth the PEG-Hb molecule and gaseous ligand molecules diffusing throughthe solvent, and 2) higher viscosity increases the shear stress of thesolution flowing against the endothelial wall, eliciting the release ofvasodilators to counteract vasoconstriction. As shown in Table 2, theviscosity of the MalPEG-Hb solution is 2.5 cPs.

Colloidal Osmotic Pressure (COP)

The COP of hemoglobin solutions containing unmodified, intra- andintermolecularly cross-linked, or PEG-surface-conjugated hemoglobin havebeen measured to determine their macromolecular solution properties(Vandegriff, K. D., R. J. Rohlfs, and R. M. Wislow. Colloid osmoticeffects of hemoglobin-based oxygen carriers. In Winslow, R. M., K. D.Vandegriff and M. Intaglia, eds, Advances in Blood SubstitutesIndustrial Opportunities and Medical Challenges. Boston, Birkhauser, pp.207-232 (1997). Tetrameric hemoglobins show nearly ideal solutionbehavior; whereas hemoglobins conjugated to PEG have significantlyhigher colloid osmotic activity and exhibit solution non-ideality(Vandegriff, K. D., M. Mcarthy, R. J. Rohls and R. M. Winslow. Colloidosmotic properties of modified hemoglobins: chemically cross-linkedversus polyethylene glycol surface conjugated. Biophys. Chem. 69: 23-30(1997). As shown in Table 2, the COP of the MalPEG-Hb solution is 50.

Stability

The stability of hemoglobin solutions containing PEG-hemoglobin havebeen determined by examining the rate of autoxidation. At roomtemperature, the autoxidation of MalPEG-Hb increased from approximately5% MetHb to 5.5% MetHb in 10 hours. The autoxidation rate for MalPEG-Hbwas therefore 0.05% per hour.

Example 4 Stability of MalPEG-Hb

The purpose of this study was to determine the stability of MalPEG-Hbduring a simulation of exemplary storage and handling conditions. Thestability during three stages of handling was assessed. Stage Irepresented the transfer from frozen storage at the production facilityto temperature conditions during shipping to the clinical site (frozenstorage study). Stage II represented the thawing of the MalPEG-Hb for 24hours to +4° C. and subsequent storage at +4° C. for five days(refrigerated study). Stage III represented the thawing of the MalPEG-Hbfor 24 hours to +4° C. and subsequent storage of MalPEG-Hb at roomtemperature for several days prior to patient administration (roomtemperature study).

Experimental Methods

Stability was defined by the rate of oxidation of the MalPEG-Hb testmaterial. The percentage of methemoglobin in the sample was measuredusing co-oximetry (IL Co-Oximeter 682, GMT, Inc., Ramsey, Minn.)Measurements were made in duplicate at each time point according to theprotocol.

Temperatures were monitored by thermometer or temperature chartrecorders. The frozen storage study was conducted over a temperaturerange of −21.0±3.0° C. The refrigerated study was conducted over atemperature range of +4.0±0.2° C. The room temperature study wasconducted over a temperature range of +21.0±1.0° C.

Temperature, total hemoglobin, and percent methemoglobin were recordedat each of the indicated time points. In the frozen and refrigeratedstudies, measurements were taken at time zero (completely thawed), onehour later, and then every 24 hours for five days. In the roomtemperature study, measurements were taken at time zero (completelythawed) and subsequently every one hour for ten hours.

Results

MalPEG-Hb exhibited no significant change in percent methemoglobinduring 6 day storage at −20° C. Similarly, MalPEG-Hb showed noappreciable change in percent methemoglobin during five day storage at+4° C. During storage at room temperature, MalPEG-Hb showed less than 1percent increase in methemoglobin over a ten hour period.

Example 5 Use of Mal-PEG to Promote Hemodynamic Stability

MalPEG-Hb was prepared as described above. A dosage of 0 (LactatedRinger's alone), 200 (“A”), 400 (“B”) or 600 mL (“C”) of MalPEG-Hb,balanced to a total volume of 1000 mL Lactated Ringer's, wasadministered to patients undergoing elective orthopedic surgery prior tospinal anesthesia induction. The results are depicted in FIG. 4.

FIG. 5 depicts the vital signs of patients that received 600 mL MalPEGHb. As shown, no elevations of blood pressure were observed compared tobaseline measurements. In contrast, detectable but slight blood pressureincreases were observed in patients that received 200 or 400 mLMalPEG-Hb.

Hypotensive events were also monitored in patients, and the results areshown below in Table 3:

TABLE 3 Ringer's 200 mL 400 mL 600 mL Lactate No. of Patients with 0 2 02 Hypotension No. of Episodes per Patient 0 2 0 3 No. of Interventions 00 0 2

As shown, the patients receiving MalPEG-Hb demonstrated less hypotensiveevents than the patients receiving the placebo.

Example 6 Expanded Clinical Study of the Use of MalPEG-Hb to EnhanceHemodynamic Stability During Surgery

MalPEG-Hb has previously been tested in a Phase I trial in healthyvolunteers and yielded no significant adverse events, and was alsoconsistent with nonclinical studies showing reduced vasoactivitycompared to stroma-free Hb. Hemodynamic instability (e.g.,cardiovascular events) measured as hypotensive events, particularly inelderly patients with cardiovascular disease, are a concern for suchpatients undergoing surgery. Considerable literature supports thehypothesis that such hypotension produces ischemia in the brain, heartand kidneys that could lead to significant postoperative morbidity. Thedevelopment of Hb-based oxygen carriers (HBOCs) to protect such surgicalpatients from the effects of these adverse events is the focus of thisexperiment.

A randomized, double-blind study of MalPEG-Hb (250 or 500 mL) and acontrol group receiving Ringer's acetate alone, was carried out at 6different hospitals in groups of 30 patients each. Consenting patientsunderwent major invasive surgery, predominantly hip replacement. Dosingoccurred prior to induction of spinal anesthesia. Safety assessmentincluded vital signs and Holter monitoring (from infusion to 24 hours),as well as clinical chemistry, coagulation, hematology, and fluidbalance. The incidence of hypotension, defined quantitatively assystolic pressure less than 90 mmHg or 75% of the baseline value, wasthe primary efficacy endpoint. Another measure of efficacy was thenecessity for pharmacologic intervention to stabilize blood pressure andfluid balance as more fully described below.

Study Population

The bulk of the patients studied in this experiment were undergoingsurgical procedures that were elective, primary hip replacements inpatients with osteoarthritis. However, a number of acute fractures werealso studied, as well as a few secondary replacements. Inclusion andexclusion criteria are presented in Table 4.

TABLE 4 Inclusion Criteria Exclusion Criteria Adult males or females(surgically sterile or Any acute or chronic condition which would limitthe postmenopausal) ASA class I-III patients aged ≧50 patient's abilityto complete the study or jeopardize the years, undergoing spinalanesthesia for acute hip safety of the patient as judged by theinvestigator fracture surgery (internal fixation or replacement) orelective hip replacement surgery Stable for surgery and anesthesia asdetermined by Patients with clinical manifestations of uncontrolledphysical examination, laboratory status, vital signs and metabolic,cardiovascular or psychiatric disorder ECG as judged by the investigatorHas been given written and verbal information and has Bloodpressure >180 mmHg (systolic) or >105 mmHg had opportunity to askquestions about the study (diastolic) measured in a supine position atscreening Patients must sign an Informed Consent Form (see Patients withrecent history of myocardial infarction or Appendix II) for the study,which has been reviewed stroke (within 6 months) and approved by theIndependent Ethics Committee (IEC) Patients with recent history ofmyocardial infarction or stroke (within 6 months) Patients with recenthistory of myocardial infarction or stroke (within 6 months) Knownalcohol or other drug dependency Patients who have received any otherinvestigational drugs within 30 days prior to administration of thestudy drug Patient on oral anti-coagulant treatment with the exceptionof low-dose (<200 mg/day) acetylsalicylic acid History or family historyof a hemoglobinopathy History of coagulopathy Professional or ancillarypersonnel involved with this studyRandomization and Blinding

The treatment assigned to each numbered patient was determined accordingto a computer generated, sequentially numbered randomization code list.The treatment groups were designated: A, 250 mL of MalPEG-Hb; B, 500 mLof MalPEG-Hb; (A and B representing the “test groups”) and C, Ringer'sacetate (representing the “placebo group”), with all administrationvolumes for each group being adjusted to the same total administrationvolume prior to administration. As MalPEG-Hb is red in color and theplacebo is transparent, efforts were also undertaken to prevent thepatient and “blinded” staff from seeing the solution being infused, i.e.the study was performed in classical “double blinded” fashion.

Materials

MalPEG-Hb was prepared essentially as described above in Examples 1 to 3with some minor variations. Approximately 24 hours beforeadministration, bottles of MalPEG-Hb were removed from the freezer (−20°C.) and placed at room temperature for gradual thawing. Ringer's acetatewas obtained from a commercial source (Fressenius Kabi A B, Uppsala,Sweden.)

Administration

Either MalPEG-Hb or placebo was administered through an establishedintravenous line via a calibrated volumetric infusion pump. A totalvolume of 1000 mL of MalPEG-Hb and/or Ringer's acetate was infused forblinding purposes before anesthesia was induced, which took place within30 minutes after the end of infusion.

Infusion of the test composition or placebo solution did not influencenormal care of the patient. Patients receiving either test or placebosolution also received any additional treatments deemed necessary forthe well being of the patient. All medical procedures and treatmentswere conducted according to the clinic's standard care. Theadministration of all continuous medication on-going at study start andconcomitant medication given during the study were noted and consideredin interpreting the results.

Pharmacokinetic Measurements and Variables

The intravascular persistence of MalPEG-Hb and intravascular productstability were determined from plasma hemoglobin and methemoglobinlevels prior to infusion, end of infusion, 6 hours after infusion, days1, 2 and 3 and 7-10 days post infusion. The results of these studieswere not out of the ordinary and are not reported herein.

Sample Collection and Analysis

Blood samples were drawn using routine methods to minimize hemolysisduring sampling and processed to insure complete separation of bloodsamples from plasma. As the plasma portion of blood samples containingMalPEG-Hb were red, it was necessary for sample processing to beperformed by an “unblended” technician. Sample analysis was performedbefore “code breaking.” Thus, the laboratory performing the analysis wasappropriately blinded.

Efficacy Measurements and Variables

The effectiveness of MalPEG-Hb as an exemplary HBOC for enhancinghemodynamic stability was studied using the following endpoints:

A. Hypotensive Episodes

The number of hypotensive episodes after infusion of MalPEG-Hb/Placebo,was defined as a systolic blood pressure (SBP)<90 mmHg or a ≧25% drop inSBP compared to pre-infusion. Each registration of a SBP fulfilling thedefinition was counted as one hypotensive episode.

B. Total Fluid Intake and Output (Fluid Balance)

Measurement were made on the day of surgery (from start of infusion to24 hours after start of infusion) and post-operative days 1, 2 and 3.Intake included: infused fluids (MalPEG-Hb/Placebo, colloids andcrystalloids), blood transfusions and oral fluid intake. Outputincluded: urine and the estimated blood loss during surgery.

The type and amount of intravenous fluids were recorded (data notshown.) The total amount of crystalloid and colloid infusions were notstatistically different among the three groups for the entirehospitalization period. However, differences did occur in theintraoperative period. Group A received significantly less crystalloid(913±106 mL) compared to Group B (1299±183 mL) and Group C (1281±144 mL)(P<0.05). Furthermore, when the MalPEG-Hb was included in the amount ofcolloid administered, Group B received significantly more total colloid(1389±169 mL) compared to Group A (850±66 mL) or Group C (666±69 mL).Both differences, B vs C and A vs B were statistically significant(P<0.05). There were no statistically significant differences incrystalloid, colloid or total intravenous fluid administration for theremainder of the study.

C. Cardiac Disturbances

The number and type of cardiac disturbances as measures of hemodynamicinstability (tachycardia, ischemia, bradycardia and conduction problems)were evaluated by the blinded cardiologist. Cardiac rhythm disturbanceswere recorded using continuous Holter-monitoring and ECG.

D. Pharmacological Interventions

The number and dose of pharmacological interventions (e.g. bloodpressure drugs, diuretics) for cardiovascular support were also recordedfrom anesthesia induction through 12 hours after start of infusion.

E. Blood Transfusions

Blood transfusions (volume of packed red blood cells) administeredduring surgery (from anesthesia induction to end of surgery andafterward) were also recorded

F. Oxygen Utilization

Duration of post-operative supplemental oxygen administration wasrecorded on the day of surgery and for a few days thereafter.

Results

Hypotensive instability manifested as adverse events, such ashypotensive episodes and the necessity for administration of pressors,are shown in Table 5 below. The results obtained for the percent ofhypotensive events are also depicted in FIG. 6.

TABLE 5 Group A Group B Group C Events (n = 29) (n = 30) (n = 31)Hypotensive events 15 15 27 % of Patents with 52 50 87 Hypotensiveevents Number of Patients 5 4 10 treated with Pressors % of Patientstreated 17 13 32 with Pressors

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the preferred embodiments of the compositions, and are notintended to limit the scope of what the inventors regard as theirinvention. Modifications of the above-described modes (for carrying outthe invention that are obvious to persons of skill in the art areintended to be within the scope of the following claims. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference as if each suchpublication, patent or patent application were specifically andindividually indicated to be incorporated herein by reference.

1. A method for reducing hypotensive events that may be experienced by anormovolemic subject during surgery, said method comprising the stepsof: a) providing a maleimide polyethylene glycol conjugated hemoglobin(MalPEG-Hb); b) monitoring blood pressure of the normovolemic subjectbefore, during and after surgery; and c) administering the MalPEG-Hb tothe normovolemic subject if the normovolemic subject exhibitshypotension; wherein administration of the MalPEG-Hb to the normovolemicsubject reduces hypotensive events.
 2. The method according to claim 1,wherein step c) is performed prior to surgery.
 3. The method accordingto claim 1, wherein step c) is performed during surgery.
 4. The methodaccording to claim 1, wherein step C) is performed after surgery.
 5. Themethod according to claim 1, wherein the polyethylene glycol (PEG) ofthe MalPEG-Hb is PEG 5000.